In scientific research, as well as in other applications, it is often desired to use multiple light sources in conducting an experiment. Multiple sources may be used either in combination in a common output beam or sequentially at different times during a research investigation (e.g., during the course of one or more experiments). The output beam can be used, for example, to illuminate an object of interest, such as a biological specimen or other object undergoing examination in a microscope. For example, two beams, each with a specific wavelength, may be simultaneously combined and used to cause excitation of specific molecules (such as florescent dyes). Beams along a common output path may be used sequentially, for example, first to set up an experiment with white light, and thereafter to use a specific light wavelength to cause an excitation. As used herein, the term “light” is intended to have broad meaning and to encompass not only visible light but also infrared and ultraviolet light. Likewise, as used herein, the term “white light” is intended to have broad meaning.
Where multiple beams are used at different times during the investigation, it is convenient for the research setup to enable the multiple beams to use a single output beam path so that it is not necessary to reconfigure the optical hardware each time a different light source is used. As used herein, the term combining beams is intended to refer to providing a common output beam path that can be used for multiple light sources either simultaneously or sequentially.
There are various sources of light, such as arc lamps, flash lamps, lasers, light emitting diodes (referred to herein as “LED's”), each of which has advantages and disadvantages in terms of power, spectral characteristics, ability to rapidly pulse, stability, etc. In many instances no one type of source provides all of the advantages that could be obtained by combining multiple sources. With improvements in LED technology, LED lamps have become increasingly useful and popular, such that there are now many specific wavelength LEDs available to the researcher. However, gaps remain in the spectrum of available LED sources, and it is often desirable to combine LEDs to obtain a desired “color.” With currently available technology, for example, white light can be made by combining three LED wavelengths (red, green and blue), and such white light may be superior for use in specific applications to the light from a “white” LED. (A “white” LED can comprise either three colored LEDs in one package or a blue LED with a phosphor excited by the blue LED, in which case some of the blue leaks through to phosphor to extend the spectral coverage. In either case the spectral output is non-uniform.)
In some cases, such as in optical microscopy systems, it is known to use moveable mirrors to switch between light sources. However, this does not allow very rapid changing of the light source, and is difficult to adapt for more than two sources of light. When multiple light sources are restricted to wavelengths that do not overlap, it is possible to combine beams efficiently using dichroic mirrors. Characteristically, dichroic mirrors reflect light above a specific wavelength while transmitting light below the specific wavelength. FIG. 1 depicts a dichroic ladder, as is known in the art, for combining multiple light beams.
FIG. 1 shows four light sources, 10a, 10b, 10c, and 10d, each of which has associated optics to collimate the light from the source into a beam, and three dichroic mirrors, 20a, 20b and 20c. The dichroic mirrors are positioned at an angle of 45° relative to the collimated beams from the light sources. First light source 10a is selected to have a wavelength which passes directly through all of the dichroic mirrors, thereby defining a combined output beam axis 5. (As shown in FIG. 1, this beam is at a right angle relative to the beams from the other light sources.) The beam from the second light source 10b has a wavelength which is reflected by dichroic mirror 20a along beam axis 5. After being reflected by mirror 20a, light from source 10b passes through the remaining mirrors 20b and 20c. Similarly, light from sources 10c and 10d are reflected by mirrors 20b and 20c, respectively, along axis 5, with the light from source 10c passing through mirror 20c. In this manner, beams from multiple sources may be combined and directed along an axis to an output (not shown).
Mirrors 20a-20c must be carefully selected and installed in the proper positions in order to allow each of the light sources to be effectively delivered to the output. In order to reconfigure the system to use different light sources, it is often necessary to reposition several of the dichroic mirrors 20 and light sources 10. Moreover, dichroic mirrors are not useful for transmitting white light since they inherently reflect all light below a cut-off frequency.
The use of interference gratings that allow only transmission of a very narrow band of light wavelengths is a well known method of filtering light. Such filters may be referred to as bandpass filters. Typically, white light is filtered to produce the desired wavelength for illuminating the object under investigation. It was common practice to combine an interference filter in series with an absorbing colored glass filter in order to improve rejection outside the desired bandpass. Advances in coating technology have allowed filters with only interference layers to achieve the same level of rejection, often with much higher transmission within the bandpass.
Bandpass filters typically reflect any light that falls out of the bandpass range. Specifically, available bandpass filters typically reflect as much as 95% of the incident light that falls outside of the narrow bandpass range, effectively acting as a mirror in respect to such wavelengths. This property could be used to combine two beams in a simplified system similar to that depicted in FIG. 1, where a single bandpass filter is used instead of a dichroic mirror. However, because of the nature of bandpass filters, no more than two beams can be combined in this manner—one with a narrow band which passes through the filter and a second which is reflected off of the filter. Moreover, usually it is best to use bandpass filters at far less than 45°, and so a tilt at this angle would not be preferred.
In many research applications, the object under investigation is very small, such that a microscope is necessary to conduct the desired experiment or procedure. In such cases, and in others, space is at a premium and it is desirable to make the various hardware systems used to conduct the investigation as compact as possible. Such hardware systems may include, for example, optical systems for illuminating a specimen, mechanical systems such as micromanipulators and the like to position the specimen, microscopy systems and probe systems for observing and making measurements of the specimen, recording systems for acquiring data and images, and control systems for operating and coordinating the hardware.
Filter wheels, useful in many applications, are well known. Basically, a filter wheel comprises a plurality of optical filters mounted on a disc-shaped “wheel” that is rotatable about a central axis. By rotating the wheel any of the filters can be positioned by the user in the light path, thereby allowing the user to select (from among the filters) the wavelength of light used to illuminate the specimen. Such filters wheels are available, for example, from Sutter Instrument Company of Novato, Calif., (www.sutter.com) assignee of the present invention.
Recently, the assignee of the present invention has developed a filter wheel incorporating filters, wherein at least one, and preferable all, of the filters on the wheel are tunable. A filter wheel using tunable bandpass filters is disclosed in the assignee's U.S. application patent Ser. No. 13/162,904, the disclosure of which is incorporated by reference.
Quite often the same microscope is used in connection with experiments involving more than one illumination and/or emission wavelength, either during a single experiment or in different experiments. Moreover, in many research applications it is beneficial to use a narrow band of filtered light at some times, while at other times illuminating the object with white light. For example, the object undergoing microscopic examination may be illuminated with white (unfiltered) light when manipulating, preparing or handling the object and, thereafter, filtered to illuminate it with one or more selected frequencies to cause characteristic emissions. As noted above, however, the space used for the investigation may be very limited, making it difficult to place and remove filters. In order to enable an experiment to proceed efficiently, it is often desirable to quickly adjust the characteristics of the illuminating beam.